The transforming growth factor (TGF-β) signaling pathway has been known to play an important role in gastrointestinal epithelial cell homeostasis; cell differentiation, proliferation, and migration; and modulation of gastrointestinal cancers (see references 2 and 15 below). The enlarging TGF-β superfamily comprises more than 40 members, which include the TGF-βs 1-3, bone morphogenetic proteins, activins, Nodal, Lefty-1, Lefty-2, anti-Müllerian hormone, and other growth/differentiation factors (24, 32, 33). Despite the diverse and complex responses they elicit, the basic signaling cascade of TGF-β is surprisingly simple and is composed of Type I and Type II transmembrane serine/threonine kinase receptors, TβRI and TβRII; the cellular response is controlled by intracellular signaling proteins, Smads (26).
Ligand binding results in phosphorylation at Gly-Ser (GS) in the cytoplasmic tail domain of TGF-β receptor type I (TβR1) by type II (TβRII), activation of Smad2, and Smad3 phosphorylation at the C-terminal serines (11, 19). Subsequent heteromeric complex formation with the L3 loop region phosphoserine-binding pockets of Smad4 facilitates nuclear translocation and TGF-β target gene activation (18, 25, 30). Adaptor proteins are required for functional specificity and Smad modulation. We have shown that ELF, a β-spectrin, is a crucial adaptor protein in TGF-β signaling, and is required for Smad3 and Smad4 localization and signaling (8, 36). This was interesting as β-spectrins are major dynamic scaffold molecules involved in generating functionally distinct membrane protein domains, conferring cell polarity, and regulating endocytic traffic (22, 35).
Originally described by its transforming capability, TGF-β is also a growth inhibitor in epithelial tissues, as it is both a suppressor and promoter of tumorigenesis. It has been suggested that nearly all colon cancers, pancreatic cancers and gastric carcinomas have mutations inactivating some component of TGF-β signaling (39, 43), from TβRII frameshift mutations with microsatellite instability (MSI), to mutations in Smad4, Smad2 or an as yet untested component of the TGF-β signaling pathway (17, 40). Genetic studies in mice have provided strong models and further evidence for the role of TGF-β in tumor suppression in early stages. Tgf-β−/−/Rag2−/− mutant mice that live to adulthood rapidly develop colon cancer by 5 months of age, preceded by precancerous lesions with inflammation and hyperplasia (16). Smad4 deficiency in the ApcΔ716 mouse increases adenoma size and promotes cancer progression (15), and Smad4−/− mutant mice develop gastric polyps and carcinomas. In addition, depending upon the genetic background of the mice, Smad3−/− mutant mice develop aggressive metastatic colorectal cancer (36). It is clear that the nature of the TGF-β signaling pathway makes it imperative to develop methods and means for examining how this pathway affects diseases of the hepatocellular and gastrointestinal organs, particularly cancer and tumor growth.
In general, the evaluation of chemical compounds for potential efficacy as human therapeutics requires data and information of a compound's efficacy which is obtained in vivo. In order to assess such compounds, it is important to utilize an animal model which most closely reflects the pathogenic conditions which the chemical compounds are being designed to treat. Traditionally, laboratory animals can be used to provide satisfactory systems for screening potential therapeutics for treating a number of human physiological disorders such as cancer drugs. Through the use of transgenic technology or directed breeding, animals can be manipulated so as to form model systems so as to study and treat a variety of disease conditions, such as U.S. Pat. No. 6,762,343, incorporated herein by reference, which relates to the study of GPX activity in the gastrointestinal tract. However, there are no current animal models which have a disruption of the TGF-β signaling pathways, and thus no current methods or models of adequately studying these pathways and their effects, including the development of tumors, or to assess drugs and other small molecules which might be used to enhance tumor suppression.
There is thus a distinct and significant need for animal models which can be utilized to study the physiological function of the proteins responsible for TGF-β signaling in the liver and gut in developing animals, including the ELF protein and the Smads, and for methods of utilizing such models to diagnose, suppress and/or treat a variety of forms of hepatocellular and gastrointestinal cancers.